Tumour therapy is mostly implemented through the use of substances targeted at destroying cancer cells. This can be achieved with cytotoxic substances, which have to penetrate into the tumour cells in order to exert their full effect, or by means of treatment of the tumour cells with radiation of sufficient energy to kill the cells. In both cases the main problem is to deliver the substance in a selective manner to the target cells, so as to avoid possible damage to the surrounding healthy cells. In the case of radiopharmaceuticals, i.e. substances carrying radioactive portions, the problem of selectively delivering the active part (that is, the radioactive portion) to the tumour target, avoiding as far as possible diffusion of the radionuclide in the body or interaction with healthy cells surrounding the tumour, is perceived as being particularly important.
For a discussion of all the issues involved and the solutions proposed to date, the reader is referred to U.S. Pat. Nos. 5,283,342, 5,608,060 and 5,955,605, assigned to NeoRx Corporation.
In these documents, the problem, amongst others, of the resistance of the molecule carrying the radionuclide to the metabolic attacks of the body is discussed. Specifically, the case most carefully studied is the molecule of biotin, which is one of the first choices for delivering the radionuclide to the tumour cells, thanks to its well-known interaction with avidins. Biotin is bound to the radionuclide-chelating portion, e.g. a molecule of tetra-azacyclododecanetetra-acetic acid [DOTA], via a linker. One of the main problems related to the use of biotin-DOTA conjugates is the resistance of the complex containing the biotin molecule, as connected to the radionuclide via the linker, to biotinidases, enzymes that break the peptide bond present in the complex. This peptide bond stems from the union of the chelating agent and biotin.
Among its much desired characteristics, this complex must be eliminated from the body rapidly and efficiently and must be sufficiently small (M.W.<1000) to allow easy distribution into the extracellular fluid where it will bind the tumour cells. In addition, it must show proven stability in vivo with only minimal uptake by non-tumour cells and rapid (renal) clearance and must not be metabolised.
Moreover, the chelating moiety must be such that it is not released in vivo, thus delivering potentially toxic compounds within the body. Experts in the field are clearly familiar with the problem of the release of radionuclide by the chelating portion, including metal ions which are entirely foreign to the body, which may be endowed with radioactivity of various types and even high-energy radiation, which is therefore highly damaging.
In a previous patent application from the same Applicant (WO 02/066075) our group reported the synthesis of a new biotin-DOTA conjugate together with binding, stability and affinity studies performed on this new derivative. The novelty of the new conjugate was that the amide carboxylic group was reduced to a methylene one thus generating the N-aminohexyl biotinamido derivative (r-BHD) in which the amide was transformed into a secondary amine without affecting the length of the biotin side-arm involved in Av/Sav binding. The DOTA ligand, in this compound, was directly linked to the amino group of the reduced biotin-hexamethylenediamine derivative through one of the four N-acetic side arms.
Moreover, the synthetic flexibility of r-BHD allows to generate a variety of new biotin derivatives for example with two DOTA chelators conjugated to the side-chain of biotin with the purpose to increase the efficacy of targeted radionuclide therapy by delivering higher radiation dose to the tumor.
US Patent application 2004/0241172, by Axworthy Donald B. et al., discloses biotin derivatives incorporating two DOTA groups, through specific linkers, which are directly bonded, through a benzyl group, to the core of DOTA molecules. This modification of the DOTA group could reduce the binding ability of the chelating moiety.
In order to improve the radiation/dose ratio we have designed and synthesized a new class of biotin derivatives.